HCV dependencies on the host machinery are both intricate and extensive. Each of these host dependencies is a potential therapeutic target. Previous efforts have been successful in discovering important steps in HCV replication, yet many fundamental processes in the viral lifecycle remain uncharacterized. Using RNAi-based genetics and an infectious HCV cell culture system, we performed an unbiased genome-wide screen to identify host factors required for productive HCV infection. We applied a two-part screening protocol to identify host factors involved in the complete viral lifecycle, from viral entry to production of infectious virus. A validation screen was subsequently performed to minimize potential off-target effects. 512 genes were identified in the initial screen and 262 were confirmed by the validation assay. We identified 238 host susceptibility factors (HSFs) and 24 host resistance factors (HRFs), the majority of which were not previously linked to HCV. Of these 262 validated hits, 45 target late-stage viral infection. Integrative bioinformatics analyses of these host genes and other published database revealed a broad and complex dependency of HCV on cellular processes and molecular functions, and also implicated novel cellular signaling pathways modulating HCV infection. Several key pathways including TGF-beta, ErbB, MAPK, focal adhesion and ubiquitin proteolysis are particularly enriched in the bioinformatics analysis. By applying various virologic assays and molecular techniques, a comprehensive map of cellular pathways and machineries that are associated with each steps of HCV lifecycle, including viral entry, intracellular trafficking, viral RNA replication and translation, polyprotein processing, virion assembly and secretion, are being established. A global identification and characterization of HCV-host interactions will significantly advance our understanding of HCV-related pathogenesis, and hence illuminates potentially valuable targets for prophylactic and therapeutic interventions. Based on the infectious HCV cell culture system, we are also setting up a cell-based assay for high-throughput screening (HTS) of small molecule chemical library in collaboration with the NIH Chemical Genomics Center. The NCGC has a large collection of over 250,000 chemical compounds and has established the facility for HTS. By performing cell-based HTS, we hope to identify novel targets and lead compounds for HCV therapeutic development. The identification of the hepatitis C virus (HCV) strain JFH-1 enabled the successful development of infectious cell culture systems. Although this strain replicates efficiently and produces infectious virus in cell culture, the replication capacity and pathogenesis in vivo are still undefined. We previously reported the in vivo phenotype of the JFH-1 virus. Cell culture-generated JFH-1 virus (JFH-1cc) and patient serum from which JFH-1 was isolated were inoculated into chimpanzees. Both animals became HCV RNA-positive 3 days after inoculation, but showed low-level viremia and no evidence of hepatitis. HCV viremia persisted 8 and 34 weeks in JFH-1cc and patient serum-infected chimpanzees, respectively. Immunological analysis revealed that HCV-specific immune responses were similarly induced in both animals. This study shows that the HCV JFH-1 strain causes attenuated infection and low pathogenicity in chimpanzees, and is capable of adapting in vivo with a unique mutation conferring enhanced replicative phenotype. As a follow-up study, we performed a comprehensive analysis of the innate and adaptive immunity following HCV re-exposure of the two chimpanzees recovered from HCV-JFH1 infection. We observed that prevention of HCV re-infection upon heterologous re-challenge depend on both the activation of intrahepatic innate and cellular immune responses. Furthermore, our results suggest that serum neutralizing antibodies may contribute to the control of viral replication and spread immediately after homologous HCV re-challenges. We conclude that protective immunity against HCV re-infection is orchestrated by a complex network of innate and adaptive immune responses.